Biomedical therapies in specific regard to tissue engineered solutions are rapidly emerging, especially as new and innovative improvements are constantly being researched and developed. An area of particular focus is bone regeneration, which has many far-reaching clinical needs. New treatment options are needed to facilitate the regeneration of lost bone and faster healing of fractures within an aging population. The present therapies include the use of allografts, autografts, and synthetic biomaterials. However, all are challenged by the variations that exist between patients, type of injury, and ability to control the biological response to ensure healing. Thus, to overcome these limitations, a biomaterial analogous to the biological and structural characteristics of natural bone, specifically the fundamental bone extracellular matrix (ECM), is needed, thereby creating a biomimetic environment with an instructive capacity for regeneration at the cellular level. Hence, this project proposes to develop a bone ECM mimicking self-assembled nanomatrix that captures the osteoinductive environment of native bone tissue at the most basic nanostructure level of tissue formation. The bone ECM consists of organic fibrous proteins and reinforcing inorganic phosphate minerals primarily composed of hydroxyapatite (HA). To recreate this basic assembly, a biphasic scaffold consisting of nanofibrous peptide amphiphiles (PAs) as the organic component and embedded inorganic HA nanoparticles will be developed to serve as a nanomatrix interface for human mesenchymal stem cells (hMSCs) isolated from bone marrow. It is hypothesized that this biphasic biomimetic scaffold will promote the enhanced osteogenic differentiation and biomineralization of hMSCs driven exclusively by the inscribed cell adhesive ligand sequences within the PAs and incorporated HA nanoparticles. Preliminary studies have already demonstrated that PA nanomatrices inscribed with specific adhesive ligands were able to direct the osteogenic differentiation of hMSCs without osteogenic supplements. Furthermore, a highly tunable composite approach for creating PA hydrogels with better regulation of the cell encapsulating microenvironments has been established by combining two functionally-specific PAs with differing mechanical properties in a controlled manner, ensuring stability and integrity across many different PA sequences. These promising results will be expanded upon in this research training plan. First, synergistic effects will be studied by including osteogenic supplements in addition to the cellular adhesive ligands and evaluating the osteogenic differentiation potential. Then, a biphasic nanomatrix will be developed by incorporating HA nanoparticles within the self-assembling PA hydrogel. The efficacy of the biphasic nanomatrix to promote the osteogenic differentiation and mineralization of hMSCs will be evaluated both in vitro and in vivo. Thus, new insights into enhanced bone regenerative biomaterials will be developed by following the principles of nature tissue formation.